The deleterious effects of tobacco smoking on human health have been extensively documented. Among other things, such conditions as cancer and coronary artery disease are dramatically elevated in incidence and severity in the case of smokers, and such patients likewise present significant alterations in lipoprotein profiles and increases in oxidized LDLs. Increased occurrence and severity of coronary artery disease and dyslipidemia, among other things, have also been observed in patients having elevated levels of advanced glycosylation endproducts (AGEs), and the coincidence of these observations set the stage for the discovery that underlies the present invention.
The nonenzymatic reactions between glucose and proteins has been recognized for many years, but molecular details of these reactions, and the biological and medical consequences of nonenzymatic glycation in vivo, are still emerging today. The earliest recognized manifestation of nonenzymatic glycation was in the appearance of brown pigments during the cooking of food, which was identified by Maillard in 1912, and lent the term "browning" to this branch of food chemistry. Maillard observed that glucose or other reducing sugars react spontaneously with amino-containing compounds such as amino acids and peptides to form initial Schiff base adducts. This condensation product then undergoes a series of additional spontaneous dehydrations, rearrangements and other reactions to form the class of brown pigments, now known as advanced glycosylation endproducts, or AGEs.
In the years that followed its initial discovery, food chemists studied the Maillard reaction in detail and determined that stored and heat-treated foods undergo nonenzymatic browning as a development of the initial reaction between glucose and the polypeptide chain, and that the proteins are resultingly crosslinked and correspondingly exhibit decreased bioavailability. At this point, it was determined that the pigments responsible for the development of the brown color in protein glycosylation (or advanced glycation) also possess characteristic absorptive spectra and fluorescent properties.
The reaction between reducing sugars and food constituents discussed above was found in recent years to have its parallel in vivo. Thus, the nonenzymatic rearrangement of the initial Schiff base formed by addition of glucose to a free amino group on a protein forms the stable amino, 1-deoxy ketosyl adduct known as the Amadori product. (A parallel reaction involving a reducing ketose rather than an aldose generates an early glycation product known as the Heyns rearrangement product). Accumulation of this early glycation adduct has been shown to occur with hemoglobin, wherein a rearrangement of the amino terminus of the .beta.-chain of hemoglobin following an initial reaction with alucose forms the modified hemoglobin known as hemoglobin A.sub.1c, a clinically important marker of glucose control in diabetes. Glycation reactions have also been found to occur with a variety of other body proteins, such as lens crystallins, collagen nerve proteins, and low density lipoproteins, as well as DNA and aminophospholipids.
The Maillard browning process generates a widely diverse array of advanced glycosylation products, each of which occurs in very low yield. This diversity has made identification and structural determination of specific AGEs a difficult proposition. In U.S. Pat. No. 4,665,192 the fluorescent chromophore 2-(2-furoyl)-4(5)-2(furanyl)-1H-imidazole was isolated and identified from certain browned polypeptides such as bovine serum albumin and poly-L-lysine. This success encouraged the subsequent identification of additional advanced glycosylation endproducts and assisted additional investigations seeking to clarify the chemistry of the protein aging process and to identify the specific reactants, intermediates and products involved in order to develop methods and agents for inhibiting glycation.
More recently, other advanced glycosylation products have been identified, such as AFGP (Farmar et al. U.S. Pat. No. 5,017,696, issued May 21, 1991); pyrraline (Hayase et al., "Aging of Proteins: Immunological Detection of a Glucose-derived Pyrrole Formed during Maillard Reaction in vivo", J. Biol. Chem., 263:3758-3764 (1989)), and pentosidine (Sell, D. and Monnier V. Structure Elucidation of a Senescence Cross-link from Human Extracellular Matrix, J. Biol. Chem., 264:21597-21602 (1989)).
A large bodv of evidence has been assembled to show that Maillard products as a whole underlie a wide variety of both normal and pathogenic activities and responses that occur as AGEs accumulate in vivo. Such activities may be direct, as a consequence of the chemical reactivity of glycation products and adducts; or indirect, mediated by the cellular recognition of glycation adducts and products via AGE-specific binding proteins or receptors.
Although most studies describing the pathogenic role of AGE accumulation in vivo have focused on AGE-proteins and AGE-peptides, the reaction between the lipids, and particularly, low-density lipoprotein (LDL) and glucose to form lipid-AGEs also has been determined to play a pathogenic role, for instance, in atherogenesis, where the formation of foam cells marks the accumulation of atherosclerotic plaques. Oxidation and glycation of the protein and lipid components of low-density lipoprotein (LDL) results in the loss of the recognition of the apo B component by cellular LDL receptors, prolonging the circulating half-life of this LDL and resulting in the preferential uptake of oxidized-LDL (ox-LDL) or otherwise modified LDL via macrophage "scavenger" receptors, AGE receptors, and other specialized cellular mechanisms. The enhanced endocytosis of ox-LDL by vascular wall macrophages has been linked to their transformation into lipid-laden foam cells that characterize early atherosclerotic lesions. Previous studies also have shown that AGE modification of LDLs increases the potential for lipid oxidation.
The "family" of AGEs includes relatively stable species which can be isolated and characterized by chemical structure, while others are unstable or reactive and their structural determination has therefore been problematic. Labile or reactive AGEs can be "trapped" by specific chemical agents, and such reactions and trapping have been used not only to gain structural insights but also to inhibit the glycation process for therapeutic purposes. AGE-lipids may also be stable, unstable or reactive.
An appreciation for the pathogenic potential of AGEs has suggested that interference with, or inhibition of, advanced glycation chemistry could be of enormous therapeutic benefit. In this connection, a series of agents has been discovered, as exemplified by aminoguanidine (also known as Pimagedine), that are useful glycation inhibitors. This compound, and others like it, have been theorized to react with the carbonyl moiety of the early glycosylation product of a target protein (or other biomolecule) formed subsequent to the initial nonenzymatic reaction with glucose or another reducing sugar, and thereby prevent further reaction to form advanced glycosylation endproducts.
A variety of other inhibitors of advanced glycation reactions are also known, many of which are thought to be particularly effective at stages of the Maillard reaction other than those which are most susceptible to inhibition by aminoguanidine and its analogs. For instance, certain compounds, or compositions thereof, having an active aldehyde substituent, such as acetaldehyde, are effective inhibitors of the advanced glycation pathway. This activity is thought to arise by the reaction of such active aldehyde agents with the glycosyl-amino moieties of glycation products formed in the initial stages of the Maillard reaction, i.e., these agents react with the Amadori and Heyns rearrangement products, which are early glycation products. Other agents that may serve in a similar capacity, are those that include a conserved binding motif which comprises a common 17-18 amino acid cysteine-bounded hydrophilic peptide loop domain, initially discovered and identified in the antibacterial proteins lysozyme and lactoferrin. More particularly, exemplary agents include molecules having an hydrophilic loop domain, which hydrophilic loop domain has a structure corresponding to R.sub.1 Z.sub.1 Xaa.sub.n Z.sub.2 R.sub.2 (SEQ ID NOS:1-6), wherein Z.sub.1 and Z.sub.2 are residues capable of forming a cross-link; R.sub.1 and R.sub.2 are independently a polypeptide, a C.sub.1 to C.sub.12 alkyl, aryl, heteroalkyl, or heteroaryl group, or hydrogen; Xaa is any L- or D-amino acid; and n=13-18. Such agents and their uses are set forth in International Publication No. WO 96/31537, published Oct. 10. 1996 incorporated herein by reference. Yet other agents such as certain thiazolium compounds, are particularly effective at preventing advanced glycation, and even reversing the formation of advanced glycation endproducts and associated cross-linking moieties, through reactions with late glycation products.
The compounds, and their compositions, utilized in this invention are thought to react with early glycosylation products, thereby preventing the same from later forming the advanced glycosylation end products which lead to cross-links, and thereby, to molecular or protein aging and other adverse molecular consequences. All of such various glycation pathway inhibitors and other compounds reactive with early and late glycation products, either alone or in combination, find use in ameliorating the pathogenic potential of AGEs that accumulate in the body.
AGEs may accumulate through de novo formation in vivo, through reattachment of AGEs liberated in vivo by cellular activities, or as revealed herein by Applicants, by exposure to exogenous AGEs, for instance by smoking. Accordingly, the significance of the reactive pathways of AGEs and their observed presence in tobacco and tobacco smoke lends further importance and encouragement to the need for the development of effective techniques for monitoring the smoking habits of individuals as well as the development of effective agents and devices to reduce the transfer of AGEs by tobacco use. Such monitoring techniques would be useful for gathering the history of such smokers, and also for monitoring their medical condition and particularly, their predisposition to the conditions that are associated with elevated levels of AGEs. Measurement of AGEs in tobacco would facilitate the evaluation of tobacco crops, as well as providing an index of the amount of AGEs that would be transferred to a smoker during the consumption of smoking materials prepared from the crops in question; in this regard, AGEs may also be evaluated in tobacco smoke. Effective therapeutic strategies, methods and agents in this regard may address the inhibition of AGEs in tobacco, in smoking materials, and would include the development of filter devices and like materials for use in conjunction with tobacco smoking, or in the protection or treatment of individuals of the smoking or smoke-exposed populations. It is toward the fulfillment of all of the above recited objectives that the present invention is directed.